Project Summary. While rates of antibiotic resistance bacterial infections continue to rise, our development of new antimicrobial agents has stagnated. The high failure rate of new candidate compounds demands the development of alternative pipelines for the discovery of new antimicrobial agents to prevent our slide back to the pre-antibiotic medical era. Our objective in this proposal is to develop and implement a new molecular approach to drug screening for an in-depth study of peptide chemistry with antimicrobial activity against antibiotic-resistant, Gram-negative bacterial pathogens. Our platform creates microenvironments for individual bacteria and peptide sequences to interact under physiologically relevant conditions, within a mixed bacterial population. Lytic events are measured using next-generation sequencing, allowing rapid and batch screening of millions of peptides in one tube. This proposal offers a quantum leap forward in antimicrobial peptide research. Completion of the planned work is expected to have a positive translational impact by greatly expanding our understanding of peptide chemistry with antimicrobial activity and identify new bacterial targets for therapeutic targeting, both of which will likely support the development of new antimicrobials and approaches to fight antibiotic-resistant bacteria.